1. Field of the Invention
The present invention is in the field of substituted 1-arylpyrazole compounds, their synthesis and their use as pest GABA receptor inhibitors and pesticides.
2. Related Art
xcex3-Aminobutyric acid (GABA) receptors are intrinsic membrane glycoproteins in vertebrate and invertebrate neuronal tissues that are members of the ligand-gated ion channel superfamily of receptors. GABA receptors play a major role in the inhibition of central nervous system (CNS) neuronal activity due to the widespread distribution of GABA-releasing and GABA-receptive neurons.
Vertebrate GABA receptors can be divided into two major classes: the GABAA and GABAC subtypes, and GABAB receptor subtype, which are distinguished by differences in their effector mechanisms and pharmacology (Knapp, R. J., et al., Neurochem. Res. 15:105-112 (1990)). GABAA and GABAC receptors are transmitter-operated chloride channels that are activated by GABA to open their chloride channel while GABAB receptors are thought to mediate changes in cyclic AMP levels through the activation of phospholipase activity (Eldefrawi, A. T. and Eldefrawi, M. E., FASEB J. 1:262-271 (1987); Knapp, R. J., et al., Neurochem. Res. 15:105-112 (1990)). The GABAA receptor and its associated chloride ion channel make up the so-called GABAA receptor-channel complex.
GABA is the endogenous ligand for the GABAA receptor of the GABAA-complex, and is the major inhibitory neurotransmitter in the vertebrate brain, in the insect CNS and at insect neuromuscular junctions (Enna et al., In: Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, Alan R. Liss, Inc., New York, pp. 41-56 (1986); Sattelle, D. B., Adv. Insect Physiol. 22:1-113 (1990)). GABA binding to its receptor stimulates chloride ion conductance through the associated chloride ion channel to inhibit synaptic transmission (Knapp, R. J., et al., Neurochem. Res. 15:105-112 (1990); U.S. Pat. No. 5,487,976). When two molecules of GABA bind at sites on the receptor, the chloride channel undergoes a conformational change and opens, allowing chloride ions to flow passively down the electrochemical gradient into the neuron. An influx of chloride into the cell causes a change in the membrane potential, usually a hyperpolarization, which results in an inhibition of the nerve impulse. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyper-excitation of the CNS, resulting in convulsions and death. In contrast, irreversible hyperactivation of the channel suppresses neuronal activity, resulting in ataxia, paralysis, coma and death (Bloomquist, J. R., Comp. Biochem. Physiol. 106C:301-314 (1993)).
GABAA receptors belong to the class 1 family of neurotransmitter/hormone receptors. Other class 1 members include the glycine receptor, the serotonin type-3 receptor, the nicotinic acetylcholine receptors (muscle and neuronal types) and several excitatory amino acid receptors of vertebrates. Class 1 receptors employ no second messengers and are found where a fast conductance is required. In contrast to class 1 receptors, class 2 receptors (e.g. muscarinic, adrenergic, and others) are coupled to a second messenger and/or a G protein for their transduction, with the channel involved being separate (and usually distant) from the receptor, which is both an agonist-binding and G protein-binding molecule (Barnard, E. A., et al., TiNS 10:502-509 (1987)).
GABAA receptors are pentameric oligomers, of about 250 kilodaltons (kDa), composed of six different types of subunits, xcex1, xcex2, xcex3, xcex4, xcex5 and xcfx81, each of approximately 50 kDa (Olsen, R. W., and Tobin, A. J., FASEB J. 4:1469-1480 (1990); Hevers, W., and Lxc3xcddens, H., Mol. Neurobiol. 18:35-86 (1998)). Each subunit comprises a large extracellular N-terminal domain that putatively includes the ligand-binding site, four hydrophobic presumed membrane-spanning domains, one or more of which contribute to the wall of the ion channel, and a small extracellular C-terminus (Lxc3xcddens, H., and Wisden, W., TiPS 12:49-51 (1991); Olsen, R. W., and Tobin, A. J., FASEB J. 4:1469-1480 (1990); Hevers, W., and Lxc3xcddens, H., Mol. Neurobiol. 18:35-86 (1998)). Heterologous expression in vitro of different combinations of GABA receptor subunit types (xcex1, xcex2, xcex3, xcex4 etc.) and subunit isoforms (xcex11, xcex12, etc. except xcex4) results in heteromultimeric receptors with differing structure and pharmacology (Schofield, P. R., TiPS 10:476-478 (1989); Burt et al., FASEB J. 5:2916-2923 (1991)).
GABA receptors also play an important role in the chemical control of pests, particularly insects, such as fleas, ticks, house flies, fruit flies, plant bugs, boll weevils, grasshoppers, cockroaches, mosquitoes, beetles, locust and moths (Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)). To date, all insect GABA receptors studied gate a fast acting chloride ion conductance. Although they appear to share many of the properties of GABAA-type receptors in the vertebrate CNS, the majority of receptors in the insect nervous system appear to be bicuculline-, pitrazepin- and RU5135-insensitive (Anthony, N. M., et al., Comp. Mol. Neurobiol., Pichon, Y., ed., Birkhxc3xa4user Verlag, Basel, Switzerland, pp. 172-209 (1993); Wafford, K. A., et al., J. Neurochem. 48:177-180 (1987)). These findings indicate that insect GABA receptors contain several drug binding sites with structural and target site specificities that are different from vertebrate receptor-binding sites (Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)). Selective insecticides, e.g. insecticides with favorable selective toxicity for insects relative to vertebrates, are based in part on this target-site specificity between the GABA receptors of insects and the GABAA receptors of vertebrates (Moffat, A. S., Science 261:550-551 (1993); Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)).
Radiolabeled ligand binding studies have considerably expanded our knowledge of insect GABA receptor pharmacology. Within the insect GABA receptor three distinct binding sites have been identified: the GABA receptor agonist binding site, a benzodiazepine binding site and a convulsant binding site (Lummis, S. C. R., Comp. Biochem. Physiol. 95C:1-8 (1990); Rauh, J. J., et al., TiPS 11:325-329 (1990)). The convulsant binding site of GABA receptors in pests is the major target site for many of the drugs and pesticides currently on the market.
Convulsant drugs and pesticides act at the GABA receptor in pest brain, ganglia and muscle as noncompetitive blockers. Inhibition of GABA receptors in pests produces neurotoxicity (e.g. convulsions, paralysis, coma and death). In the early 1980s, the pesticides lindane and cyclodienes (e.g. dieldrin) were shown to antagonize the action of GABA in stimulating chloride uptake by various pest nerve and muscle preparations (Narahashi, T., Pharmacol. Toxicol. 78:1-14 (1996)). GABA receptors in pests are also blocked by picrotoxin, phenylpyrazole pesticides (e.g. Fipronil(copyright)), bicyclophosphorous esters (e.g. t-butylbicyclophosphoronthionate), and bicycloorthobenzoates (4-n-propyl-4xe2x80x2-ethynylbicycloorthobenzoate) (U.S. Pat. No. 5,853,002). These pesticides block transmission of signals by GABA, and are very effective on a wide range of economically important pests.
Unfortunately, many potent pesticides and their derivatives also act at the GABAA receptors of animals. For example, fipronil sulfone and desulfinyl fipronil, a metabolite and photoproduct of fipronil, respectively, are not only toxic to pests, but also to upland game birds, freshwater fish and invertebrates, and waterfowl. In addition, fipronil itself is a toxicant for mammals even without oxidation to the sulfone (Hainzl, D., et al., Chem. Res. Toxicol. 11:1529-1535 (1998)).
Pesticides that effectively kill pests but that have little toxicity for animals and humans remain the aim of current research efforts. The present invention addresses the need for the development and use of new and more efficacious pesticides that are highly toxic to pests but not to animals susceptible to pest infestation.
A first aspect of the present invention is directed to novel compounds of Formula I.
A second aspect of the present invention is directed to pesticidal compositions comprising at least one compound of Formula I, or a salt thereof, and one or more pesticidally-acceptable excipients.
A third aspect of the present invention is directed to a method of inhibiting a pest GABA receptor, comprising contacting one or more pest GABA receptors with one or more compounds of Formula I.
A fourth aspect of the invention is directed to a method for controlling pests, comprising contacting an animal, plant or object with a composition comprising a pesticidally effective amount of at least one compound of Formula I, or a salt thereof, and one or more pesticidally-acceptable excipients.
A fifth aspect of the present invention is directed to a method for synthesizing compounds of Formula I.
A sixth aspect of the invention is directed to the use of one or more compounds of Formula I for the manufacture of collars or external devices, as well as to a treatment process relating thereto.
A first aspect of the invention is directed to compounds of Formula I. Such compounds include compounds of Formula I: 
or a salt thereof, wherein
R1 is amino, hydrogen, alkyl, hydroxy, halo, alkoxy, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, trifluoroalkylsulfinyl, trifluoroalkylsulfonyl, hydroxy, trifluoromethyl, acetylamino, xe2x80x94OSO2R2, xe2x80x94OS(O)R2, xe2x80x94OC(O)R2, xe2x80x94OC(O)NHR2, or xe2x80x94NHC(O)NHR2 where R2 is C1-4 alkyl or optionally substituted aryl;
X, Y and Z are each independently (CH)n, (CR3R4)n, S, S(O), or SO2, wherein n is 1-2;
Q is N or Cxe2x80x94R6, wherein R6 is fluoro, chloro, bromo, or iodo;
R5 is halo, C1-C6alkyl, C1-C4 alkenyl, C1-C4 alkoxy, C1-C4 alkylamino, C1-C4 dialkylamino, C1-C4 alkylthio, C1-C4 alkylsulfinyl, C1-C4 alkylsulfonyl, C1-C4 trifluoroalkylsulfinyl, C1-C4 trifluoroalkylsulfonyl, hydroxy, amino, or trifluoromethyl;
R3 and R4 are each independently hydrogen, halo, alkyl, alkenyl, alkynyl, cycloalkyl, cyano, trifluoromethyl, aryl, alkylamino, dialkylamino, alkoxy, cycloalkoxy, trifluoroalkyl, pentafluoroalkyl, perfluoroalkyl, thioalkyl, cycloalkylthio, trifluoroalkylthio, alkylthio, alkylsulfinyl, alkylsulfonyl, amino, or hydroxy,
or R3 and R4 taken together are oxo,
or R3 and R4 taken together with the carbon to which they are attached form a 3- to 7-membered saturated ring optionally including one or two oxygen or sulfur atoms, said ring being optionally substituted by one to three C1-4 alkyl groups;
or vicinal R4 can form an optionally substituted cycloalkyl or aryl ring while R3 is as defined above;
with the proviso that only one of X, Y and Z can be S, S(O) or SO2.
Preferred values of Q are Cxe2x80x94R6 wherein R6 is fluoro, chloro, bromo, or iodo. The most preferred value of Q is Cxe2x80x94R6 wherein R6 is chloro.
Preferred values of R5 are CF3 and Cl. The most preferred value of R5 is CF3.
A second aspect of the invention is directed to pesticidal compositions comprising a pesticidally effective amount of at least one compound of Formula I, or a pesticidally-acceptable salt thereof, wherein R1-R6, Q, X, Y, and Z are as defined above, and one or more pesticidally-acceptable excipients.
A third aspect of the present invention is directed to a method of inhibiting a pest GABA receptor, comprising contacting one or more pest GABA receptors with one or more compounds of Formula I or a pesticidally-acceptable salt thereof, wherein R1-R6, Q, X, Y, and Z are as defined above.
A fourth aspect of the present invention is directed to methods for controlling pests, comprising contacting an animal, plant or object with a composition comprising a pesticidally effective amount of at least one compound of Formula I, or a pesticidally-acceptable salt thereof, wherein R1-R6, Q, X, Y, and Z are as defined above, and one or more pesticidally-acceptable excipients. For purposes of the present invention, pests are undesired arthropods, in particular insects and arachnids, which are harmful to plants or animals susceptible to infestation by such arthropods. The methods of the present invention are suitable for combating animal pests, preferably arthropods, in particular insects and arachnids, encountered in and on companion animals, in agriculture, in forestry, in the protection of stored products and of materials, and in the hygiene field. Compounds employed in the methods of the invention have good plant tolerance or favorable safety to warm-blooded animals.
In particular, compounds of Formula I may be applied to control arthropods in compositions suitable for internal or external administration to vertebrates, or application for the control of arthropods in any indoor or outdoor area. Such compositions comprise at least one compound of Formula I and one or more excipients. The methods are more preferably used to reduce the viability and/or reproductive capacity of any ectoparasite. Preferred ectoparasites to target include arachnids, insects and leeches. More preferred ectoparasites include fleas and ticks. For example, the invention can be employed for killing fleas of the genus Ctenocephalides, in particular C. felis and C. canis, and ticks, in particular of the genus Rhipicephalus, especially R. sanguineus, as well as harvest ticks (Trombicula automnalis), Dermacentor variabilis, Dermacentor andersoni, Dermacentor occidentalis, Amblyomma americanum, Ixodes scapularis, and Ixodes pacificus. 
One subclass of compounds and pharmaceutically acceptable salts thereof include compounds of Formula II: 
or a salt thereof, wherein
R1 is amino, hydrogen, alkyl, hydroxy, halo, alkoxy, acetylamino, xe2x80x94OSO2R2, xe2x80x94OS(O)R2, xe2x80x94OC(O)R2, xe2x80x94OC(O)NHR2, or xe2x80x94NHC(O)NHR2 where R2 is C1-4 alkyl or optionally substituted aryl;
X, Y and Z are each independently (CH)n, (CR3R4)n, S, S(O), or SO2, wherein n is 1-2;
R3 and R4 are each independently hydrogen, halo, alkyl, alkenyl, alkynyl, cycloalkyl, cyano, trifluoromethyl, aryl, alkoxy, cycloalkoxy, trifluoroalkyl, pentafluoroalkyl, perfluoroalkyl, thioalkyl, cycloalkylthio, trifluoroalkylthio, alkylthio, alkylsulfinyl, alkylsulfonyl or hydroxy,
or R3 and R4 taken together are oxo,
or R3 and R4 taken together with the carbon to which they are attached form a 3- to 7-membered saturated ring optionally including one or two oxygen or sulfur atoms, said ring being optionally substituted by one to three C1-4 alkyl groups;
or vicinal R4 can form an optionally substituted cycloalkyl or aryl ring while R3 is as defined above;
with the proviso that only one of X, Y and Z can be S, S(O) or SO2.
A second subclass of compounds and pharmaceutically acceptable salts thereof are compounds of Formula I wherein R3 and R4 are each independently hydrogen, halo, alkyl, cyano, trifluoromethyl, aryl, alkoxy or hydroxy, or R3 and R4 taken together are oxo or R3 and R4 taken together with the carbon to which they are attached form a 3- to 7-membered saturated ring optionally including one or two oxygen or sulfur atoms, said ring being optionally substituted by 1-3 C1-4 alkyl groups; or vicinal R4 can form an optionally substituted cycloalkyl or aryl ring.
Preferred values of R3 and R4 include hydrogen; C1-4 alkyl including methyl, ethyl, n-propyl, i-propyl, butyl, t-butyl and i-butyl; fluoro; bromo; cyano; trifluoromethyl; phenyl; C1-4 alkoxy including methoxy and ethoxy; and hydroxy. Preferred values of R3 and R4 when taken together include oxo, ethylenedithio and optionally substituted alkylenedioxy such as ethylenedioxy and propylenedioxy.
Preferred R1 include hydrogen, C6-10 arylsulfonyl, methyl, methoxy, hydroxy, acetylamino, amino, benzoyl, chloro and bromo. Most preferably, R1 is hydrogen, amino and acetylamino.
Preferred values of X, Y and Z are such that, taken together, X, Y and Z form xe2x80x94CHxe2x95x90CHxe2x80x94Sxe2x80x94, xe2x80x94CH2SCH2xe2x80x94, xe2x80x94(CH2)2Sxe2x80x94, xe2x80x94CH2S(O)CH2xe2x80x94, xe2x80x94CH2SO2CH2xe2x80x94, xe2x80x94CH2CH2SCH2xe2x80x94, xe2x80x94CH2CH2S(O)CH2xe2x80x94, xe2x80x94CH2CH2SO2CH2xe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94(CH2)4xe2x80x94, xe2x80x94(CH2)5xe2x80x94, xe2x80x94(CF2)3xe2x80x94 xe2x80x94C(CH3)2CH2CH2xe2x80x94, xe2x80x94C(CH3)2CH2CH2CH2xe2x80x94, xe2x80x94CH2CF2Sxe2x80x94, xe2x80x94CH2CF2SO2xe2x80x94, xe2x80x94CH2CF2S(O)xe2x80x94, 
Most preferred values of X, Y and Z include xe2x80x94CH2SCH2xe2x80x94, xe2x80x94CH2S(O)CH2xe2x80x94, xe2x80x94CH2SO2CH2xe2x80x94 and xe2x80x94(CH2)3xe2x80x94.
A third subclass of compounds and pharmaceutically acceptable salts thereof includes compounds of Formula II, or a salt thereof, wherein
R1 is hydrogen;
X, Y, and Z are each independently (CH)n, or (CR3R4)n, wherein n is 1-2 and R3 and R4 are each independently:
hydrogen; or
hydroxy; or
fluoro, chloro, bromo or iodo; or
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C7 cycloalkyl; or
C1-C6 alkoxy, C3-C7 cycloalkyloxy, trifluoroalkyl, pentafluoroalkyl, or perfluoroalkyl; or
C1-C6 thioalkyl, C3-C7 cycloalkylthio, C1-C2 trifluoroalkylthio or C1-C6 alkylthio; or
C1-C6 sulfinyl, C1-C6 sulfonyl, C1-2 trifluoroalkylsulfinyl, C1-2 trifluoroalkylsulfonyl; or
R3 and R4 are taken together form oxo.
In said third subclass of compounds, preferred values of R1 include hydrogen, C1-4 alkyl, amino, and C1-4 alkoxy, more preferably hydrogen.
Preferred values of X, Y and Z are such that, taken together, X, Y and Z form xe2x80x94CH2CH2CR3R4xe2x80x94, xe2x80x94CH2CR3R4CH2xe2x80x94, xe2x80x94CR3R4CH2CH2xe2x80x94, xe2x80x94CR3R4CH2CR3R4xe2x80x94, xe2x80x94CR3R4CR3R4CH2xe2x80x94, xe2x80x94CH2CR3R4CR3R4xe2x80x94, and xe2x80x94CR3R4CR3R4CR3R4xe2x80x94.
Preferred values of R3 and R4 are hydrogen, hydroxy, fluoro, chloro, bromo or iodo; C1-C6 alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C7 cycloalkyl; C1-C6 alkoxy, C2, C3-C7 cycloalkyloxy; trifluoroalkyl, pentafluoroalkyl, perfluoroalkyl; C1-C6 thioalkyl, C3-C7 cycloalkylthio, C1-C2 trifluoroalkylthio, C1-C6 alkylthio; C1-C6 sulfinyl, C1-C6 sulfonyl, C1-2 trifluoroalkylsulfinyl, or C1-2 trifluoroalkylsulfonyl. Preferred values of R3 and R4 when taken together include oxo, ethylenedithio and optionally substituted alkylenedioxy such as ethylenedioxy and propylenedioxy.
More preferred values of R3 and R4 are hydrogen, fluoro, chloro, methyl, ethyl, hydroxy, methoxy, trifluoromethoxy, methylthio, trifluoromethylthio, methylsulfinyl, methylsulfonyl, trifluoromethylsulfinyl, and trifluoromethylsulfonyl. More preferred values of R3 and R4 when taken together are oxo, ethylenedithio, ethylenedioxy, and propylenedioxy.
A fourth subclass of compounds and pharmaceutically acceptable salts thereof are compounds of Formula II wherein:
R1 is hydrogen;
X, Y and Z are each independently (CH)n or (CR3R4)n, wherein n is 1-2;
R3 and R4 are each independently hydrogen, fluoro, hydroxy, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C3-C7 cycloalkyl; or
R3 and R4 are taken together form oxo.
Examples of suitable compounds include:
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno[3,4-c]pyrazole-3-ylamine (1);
3-amino-2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno-[3,4-c]pyrazole-5-one (1xe2x80x2);
N-{2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno[3,4-c]pyrazol-3-yl}acetamide (2);
N-{2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5-oxo-4,6-dihydrothieno-[3,4-c]pyrazol-3-yl}acetamide (2xe2x80x2);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno-[3,4-c]pyrazole (3);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno-[3,4-c]pyrazole-5-one ((xc2x1), (+), (xe2x88x92)-3xe2x80x2);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno-[3,4-c]pyrazole-5,5-dione (3xe2x80x3);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrothieno-[3,4-c]pyrazole-3-yl benzenesulfonate (6);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2H,4H,6H,7H-thiano[4,3-c]pyrazole (10);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (11);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-6,6-dimethyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (12);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,5,6,7-tetrahydro-2H-indazole (13);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole (15);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]indeno[1,2-c]pyrazole (16);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2H-thieno[3,2-c]pyrazole (22);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2H-indazole (46);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-3-methyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (33);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-3-methyl-4,5,6,7-tetrahydro-2H-indazole (34);
3,4-diaza-4-[2,6-dichloro-4-(trifluoromethyl)phenyl]tricyclo[5.2.1.02.6]deca-2,5-diene (35);
8-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5,9-trimethylspiro-[1,3-dioxane]-2,5xe2x80x2-2,4xe2x80x2,5,6xe2x80x2,7xe2x80x2-tetrahydro-2H-indazole (36);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2,4,6,7-tetrahydroindazole-5-one (37);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5-phenyl-4,5,6,7-tetrahydro-2H-indazole (39);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6,6-trimethyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (45);
5,6-diaza-5-[2,6-dichloro-4-(trifluoromethyl)phenyl]tetracyclo-[8.2.1.02, 90.03,7]trideca-3,6-diene (47);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4-dimethyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (38);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-phenyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (40);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-methyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (42);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-hydroxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (62);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-methoxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (71);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-trifluoromethoxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-methylthio-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (68);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-trifluoromethylthio-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (65);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-hydroxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-methoxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-trifluoromethoxy-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-methylthio-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-trifluoromethylthio-2,4,5,6-trihydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-6,6-difluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (50);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4-difluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (61);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole;
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4,6,6-tetrafluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (53);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4,5-trifluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (56);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4,5,5-tetrafluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (92);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-4-methyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-4-ol (93);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-5,6-dihydrocyclopenta[c]pyrazol-4-one (91);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,5-difluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (90);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,6-dihydrocyclopenta[c]pyrazol-5-one (85);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4,4,5,6-tetrafluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (89);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,6-difluoro-4-methyl-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-4-ol (88);
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,6-difluoro-5,6-dihydrocyclopenta[c]pyrazol-4-one (87); and
2-[2,6-dichloro-4-(trifluoromethyl)phenyl]-5,6-difluoro-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (86);
The term xe2x80x9coptionally substitutedxe2x80x9d when not otherwise explicitly provided for refers to the replacement of a hydrogen (or in the case of keto, two hydrogens) in a particular radical, with a functional group selected from the group consisting of halogen, trifluoromethyl, hydroxy, C1-4 alkoxy, C1-4 alkylthio, C1-4 alkylsulfinyl, amino, nitro, cyano, C2-6 carboxyalkyl, amidine, tetrazolyl, mono- or di-(C1-6) alkylamino, mono- or di-(C6-10) arylamino, C6-10 arylthio, C6-10 arylsulfonyl, C6-10 arylsulfinyl, keto, C6-10 aryl hydrazone, aminocarbonyl, mono- or di-(C1-6) alkylaminocarbonyl and mono- or di-(C1-6) alkylamino-thiocarbonyl.
The term xe2x80x9calkylxe2x80x9d as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 10 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, and decyl. Preferably, the alkyl chain is 1 to 8 carbon atoms in length, more preferably from 1 to 4 carbon atoms in length. The term xe2x80x9ccycloalkylxe2x80x9d includes bicycloalkyl and other bridged ring structures.
The term xe2x80x9carylxe2x80x9d as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 14 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl, tetrahydronaphthyl, or biphenyl.
The terms xe2x80x9calkoxyxe2x80x9d refers to any of the above alkyl groups linked to an oxygen atom.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine or fluorine being preferred.
The term xe2x80x9cvicinalxe2x80x9d is used herein to refer to substituents on adjacent carbon atoms. For example, reference to xe2x80x9cvicinal R4xe2x80x9d denotes both R4 substituents in a xe2x80x94CR3R4xe2x80x94CR3R4xe2x80x94 group.
By the term xe2x80x9cpesticidally-acceptable saltsxe2x80x9d is meant salts the cations of which are known and accepted in the art for the formation of salts of pesticidally active acids for agricultural or horticultural use. When intended for application to vertebrates to combat infection or infestation by arthropods, the salts with bases used will be non-toxic to vertebrates. By the term xe2x80x9cnon-toxicxe2x80x9d is meant salts with bases the cations of which are innocuous to the vertebrates at the doses administered and which do not vitiate the beneficial effects produced by the anion.
Preferably, the salts are water-soluble. Suitable salts with bases include alkali metal (e.g. sodium and potassium), alkaline earth metal (e.g. calcium and magnesium), ammonium and amine (e.g. diethanolamine, triethanolamine, octylamine, morpholine and dioctylmethylamine) salts. Where reference is made in the present specification to the compounds of Formula I such reference is intended to include also the salts with pesticidally-acceptable bases of compounds of Formula I where appropriate.
As used herein, the term xe2x80x9cexcipientxe2x80x9d refers to the additives used to convert pesticidally-active compounds into forms suitable for their intended purpose. For pesticidal compositions of the present invention suitable for administration to an animal, the term xe2x80x9cexcipientxe2x80x9d is meant to include, but not be limited by, those excipients described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 2nd Edition (1994), which is herein incorporated by reference in its entirety. The term xe2x80x9cexcipientsxe2x80x9d is meant to include diluents, carriers, fillers, binders, disintegrating agents, lubricants, coatings, solvents, suspending agents, dyes, extenders, surfactants, auxiliaries and the like as understood by those of skill in the art.
The term xe2x80x9cpesticidally-acceptable excipientxe2x80x9d is meant to include excipients which are known and accepted in the art for the formation of pesticides for agricultural or horticultural use. When intended for application to vertebrates to combat infection or infestation by arthropods, the excipients used will be non-toxic to vertebrates. The term xe2x80x9cnon-toxicxe2x80x9d is meant to refer to excipients which are innocuous to the vertebrates at the concentrations administered and which do not vitiate the beneficial effects produced by the active pesticide.
The compounds of Formula I can be employed as pesticides. For purposes of the present invention, pests are undesired arthropods, for example insects or arachnids, which are harmful to plants or animals susceptible to infestation by such arthropods.
Compounds of the invention are suitable for controlling animal pests, preferably arthropods, in particular insects and arachnids, encountered in and on companion animals, in agriculture, in forestry, in the protection of stored products and of materials, and in the hygiene field, and have good plant tolerance or favorable safety to warm-blooded animals.
Compounds of the invention while active against plant, hygiene and stored product pests, are particularly useful in the veterinary medicine sector, against animal ectoparasites such as scaly ticks, argasidae, scab mites, trombidae, flies (stinging and sucking), parasitic fly larvae, lice, hair lice, bird lice and fleas. For example, the compounds have activity against fleas, such as fleas of the genus Ctenocephalides, in particular C. felis and C. canis, and ticks, such as ticks of the genus Rhipicephalus, especially R. sanguineus, as well as harvest ticks (Trombicula automnalis), Dermacentor variabilis, Dermacentor andersoni, Dermacentor occidentalis, Amblyomma americanum, Ixodes scapularis, and Ixodes pacificus. By virtue of their activity against fleas and ticks, compounds of the invention are suitable for treating companion animals, such as dogs and cats.
Compounds of the invention are also suitable for the controlling of arthropods which infest useful animals in agriculture such as, cattle, sheep, goats, horses, pigs, donkeys, camels, buffalo, rabbits, hens, turkeys, ducks, geese, bees, other domestic animals such as, dogs, cats, cage birds, aquarium fish and so-called experimental animals such as, hamsters, rabbits, guinea pigs, rats and mice. The aim of combating these arthropods is to reduce fatalities and reductions in yield (in meat, milk, wool, skins, eggs, honey, etc.) so that the use of a compound according to the invention renders the keeping of animals more economic and more simple.
Compositions and methods of the present invention can be used to reduce the viability and/or reproductive capacity of any ectoparasite. Preferred ectoparasites to target include arachnids, insects and leeches. More preferred ectoparasites include fleas; ticks, including both hard ticks of the family Ixodidae (e.g., Ixodes and Amblyomma) and soft ticks of the family Argasidae (e.g., Ornithodoros, such as O. parkeri and O. turicata); flies, such as midges (e.g., Culicoides), mosquitos, houseflies, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders; lice; mites; and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas disease. Even more preferred ectoparasites include fleas, mosquitos, midges, houseflies, sandflies, blackflies, ticks and kissing bugs, with fleas, ticks, mosquitos, houseflies and midges being even more preferred.
Particularly preferred compositions and methods of the present invention targets fleas. Preferred fleas include Ctenocephalides, Xenopsylla, Pulex, Tunga, Nosopsyllus, Diamanus, Ctopsyllus and Echidnophaga fleas, with Ctenocephalides canis and Ctenocephalides felis fleas being even more preferred. For the purposes of illustration, many of the following embodiments discuss efficacy against fleas. Such discussion of efficacy against fleas is not intended, in any way, to limit the scope of the present invention.
A preferred aspect of the invention is directed towards killing fleas of the genus Ctenocephalides, in particular C. felis and C. canis, and ticks, in particular of the genus Rhipicephalus, especially R. sanguineus, as well as harvest ticks (Trombicula automnalis), Dermacentor variabilis, Dermacentor andersoni, Dermacentor occidentalis, Amblyomma americanum, Ixodes scapularis, and Ixodes pacificus. 
A further aspect of the present invention is the use of a compound of Formula I for the production of a collar or other external device intended to be attached or attachable to an animal, in particular cats and dogs.
This aspect of the invention is directed mainly towards fleas of the genus Ctenocephalides, in particular C. felis and C. canis, and ticks, in particular of the genus Rhipicephalus, especially R. sanguineus, as well as harvest ticks (Trombicula automnalis), Dermacentor variabilis, Dermacentor andersoni, Dermacentor occidentalis, Amblyomma americanum, Ixodes scapularis, and Ixodes pacificus. 
Collars intended to eliminate common ectoparasites from cats and dogs consist of a matrix, usually a plastic matrix, which incorporates a compound of Formula I, preferably between 5 and 40% active substance, and is capable of releasing the compound over time.
Slow release compositions that can be in the form of a collar or earrings for controlling harmful insects are also contemplated. Such formulations comprise from about 0.5 to about 25% active material, from about 75 to about 99.5% of a suitable resin, such as polyvinyl chloride and a catalytic amount of a plasticizer, such as dioctyl phthalate.
A subject of the present invention is thus a collar or other external device for a pet, in particular a cat or dog, made of a matrix in which is incorporated from about 0.1 to about 40% by weight, relative to the collar, of a substance which is active against ectoparasites such as fleas and ticks (anti-flea and anti-tick collar or other external device), this active substance being formed of at least one compound corresponding to Formula I.
One aspect of this method is non-therapeutic and in particular relates to the cleaning of animal hairs and skin by elimination of the parasites which are present, as well as their residues and secretions. The treated animals thus have hair which is more pleasant to look at and to feel.
The invention also relates to such a method for therapeutic purposes, intended to treat and prevent parasitoses having pathogenic consequences.
Compounds of Formula I may be applied to control arthropods in compositions suitable for internal or external administration to vertebrates or application for the control of arthropods in any indoor or outdoor area. Such compositions comprise at least one compound of Formula I and one or more excipients. Such compositions can be prepared in any manner known in the art.
Suitable means of applying compounds of Formula I include:
to persons or animals infested by or exposed to infestation by arthropods by parenteral, oral or-topical application. Examples include incorporation of an active compound in feed or suitable orally-ingestible pharmaceutical formulations, edible baits, salt licks, dietary supplements, pour-on and spot-on formulations, sprays, baths, dips, showers, jets, dusts, greases, shampoos, creams, wax-smears and livestock self-treatment systems; to the environment in general or to specific locations where pests may lurk, including stored products, timber, household goods, and domestic and industrial premises, as sprays, fogs, dusts, smokes, wax-smears, lacquers, granules and baits, and in tricklefeeds to waterways, wells, reservoirs and other running or standing water; to domestic animals in feed to control fly larvae feeding in their feces;
to growing crops as foliar sprays, dusts, granules, fogs and foams; also as suspensions of finely divided and encapsulated compounds of Formula I;
as soil and root treatments by liquid drenches, dusts, granules, smokes and foams; and
as seed dressings by liquid slurries and dusts.
Compositions suitable for administration to vertebrates include preparations suitable for oral, parenteral, percutaneous, e.g. pour-on, spot-on or other topical administration.
Compositions for oral administration comprise one or more of the compounds of Formula I in association with non-toxic veterinary carriers or coatings and include, for example, chewable treats, tablets, pills, capsules, pastes, gels, drenches, medicated feeds, medicated drinking water, medicated dietary supplements, slow-release boluses or other slow-release devices intended to be retained within the gastro-intestinal tract. Any of these may incorporate active ingredient contained within microcapsules or coated with acid-labile or alkali-labile or other pharmaceutically acceptable enteric coatings. Feed premixes and concentrates containing compounds of the present invention for use in preparation of medicated diets, drinking water or other materials for consumption by animals may also be used.
Compositions for parenteral administration include solutions, emulsions or suspensions in any suitable veterinary vehicle and solid or semisolid subcutaneous implants or pellets designed to release active ingredient over a protracted period and may be prepared and made sterile in any appropriate manner known to the art.
Compositions for percutaneous and topical administration include sprays, dusts, baths, dips, showers,jets, greases, shampoos, creams, wax-smears, or spot-on or pour-on preparations. Compounds of Formula I can also be administered with the aid of shaped articles which contain active compound, such as neck bands, ear tags, tail tags, limb bands, halters, marking devices and the like devices (e.g. ear tags) attached externally to animals in such a way as to provide local or systemic arthropod control.
Solid or liquid baits suitable for controlling arthropods comprise one or more compounds of Formula I and a carrier or diluent which may include a food substance or some other substance to induce consumption by the arthropod.
Medicated feeds which comprise a compound of Formula I and arthropodicidally-acceptable salts thereof and an edible carrier or diluent form an additional feature of the present invention.
Liquid compositions include water miscible concentrates, emulsifiable concentrates, flowable suspensions, wettable or soluble powders containing one or more compounds of Formula I which may be used to treat substrates or sites infested or liable to infestation by arthropods including premises, outdoor or indoor storage or processing areas, containers or equipment and standing or running water.
Solid homogenous or heterogenous compositions containing one or more compounds of Formula I, for example granules, pellets, briquettes or capsules, may be used to treat standing or running water over a period of time. A similar effect may be achieved using trickle or intermittent feeds of water dispersible concentrates.
Compositions in the form of aerosols and aqueous or non-aqueous solutions or dispersions suitable for spraying, fogging and low- or ultra-low volume spraying may also be used.
The compositions of the invention, besides at least one compound of Formula I and, if appropriate, besides extenders and auxiliaries, may also comprise at least one surfactant (wetting, dispersing and emulsifying agents).
The wetting, dispersing and emulsifying agents which may be present, particularly in wettable powders, may be of the ionic or non-ionic types, for example sulphoricinoleates, quaternary ammonium derivatives or products based upon condensates of ethylene oxide with nonyl- and octylphenol, or carboxylic acid esters of anhydrosorbitols which have been rendered soluble by etherification of the free hydroxy groups by condensation with ethylene oxide, or mixtures of these types of agents. Wettable powders may be treated with water immediately before use to give suspensions ready for application.
Liquid compositions for the application of the compounds of Formula I may take the form of solutions, suspensions and emulsions of the compounds of Formula I optionally encapsulated in natural or synthetic polymers, and may, if desired, incorporate wetting, dispersing or emulsifying agents. These emulsions, suspensions and solutions may be prepared using aqueous, organic or aqueous-organic diluents, for example acetophenone, isophorone, toluene, xylene, mineral, animal or vegetable oils, and water soluble polymers (and mixtures of these diluents), which may contain wetting, dispersing or emulsifying agents of the ionic or non-ionic types or mixtures thereof, for example those of the types described above. When desired, the emulsions containing the compounds of Formula I may be used in the form of self-emulsifying concentrates containing the active substance dissolved in the emulsifying agents or in solvents containing emulsifying agents compatible with the active substance, the simple addition of water to such concentrates producing compositions ready for use.
Compositions containing compounds of Formula I which may be applied to control arthropod pests, may also contain synergists (e.g. piperonyl butoxide or sesamex), stabilizing substances, other insecticides or other pesticides, acaricides, plant nematocides, anthelmintics or anticoccidials, fungicides (agricultural or veterinary as appropriate, e.g. benomyl, iprodione), bactericide, antivirals, arthropod or vertebrate attractants or repellents or pheromones, reodorants, flavoring agents, dyes and auxiliary therapeutic agents, e.g. trace elements or vitamins. These may be designed to improve potency, persistence, safety, uptake where desired, spectrum of pests controlled or to enable the composition to perform other useful functions in the same animal or area treated.
Examples of other pesticidally-active compounds which may be included in, or used in conjunction with, the compositions of the present invention are: chlorpyrifos, demeton-S-methyl, disulfoton, ethoprofos, fenitrothion, malathion, parathion, triazophos, amitraz, cypermethrin, deltamethrin, fenpropathrin, fenvalerate, permethrin, aldicarb, carbosulfan, pirimicarb, bendiocarb, teflubenzuron, dicofol, endosulfan, lindane, benzoximate, avermectins, ivermectin, milbemycins, thiophanate, trichlorfon, dichlorvos, diaveridine and dimetridazole.
A xe2x80x9cpesticidally effective amountxe2x80x9d refers to an amount of compound that will be toxic to one or more pests under the conditions administered. When administered to vertebrates parenterally, orally or by percutaneous or other means, the dosage of compounds of Formula I will depend upon the species, age and health of the vertebrate and upon the nature and degree of the vertebrate""s actual or potential infestation by arthropod pest. Determination of optimal ranges of effective amounts of each component in a composition is within the skill of the art. A single dose of 0.1 to 100 mg, preferably 2.0 to 20.0 mg, per kg body weight of the animal per month or doses of 0.01 to 20.0 mg, preferably 0.1 to 5.0 mg, per kg body weight of the animal per day for sustained medication are generally suitable by oral, topical or parenteral administration. By use of sustained release formulations or devices, the daily doses required over a period of months may be combined and administered to animals on a single occasion.
Compounds are screened for GABA receptor inhibiting activity using in vitro assays that measure the ability of a test compound to bind to pest and/or mammal GABA receptors. These assays, exemplified herein in working Examples 62 and 63, employ membranes possessing active GABA receptors. Preferred compounds have selectivity towards arthropod GABA receptor versus mammalian GABA receptor. Immediately following is a description of methods for forming such membranes. Ectoparacitidal activity can be determined in vivo. Suitable tests are described in working Examples 64 and 65.
Preparation of Housefly Membranes Possessing Active GABA Receptors
Newly emerged houseflies (Musca domestica, available from Rincon-Vitova Insectaries, Inc., Ventura, Calif.) were sedated with carbon dioxide gas, collected in 50 mL polypropylene conical tubes, and immediately frozen by submersion in liquid nitrogen. Unless specified, all of the following work was performed at 0-4xc2x0 C. After removal from liquid nitrogen, the tubes of frozen houseflies were shaken vigorously by hand to decapitate the houseflies. The decapitated houseflies were then passed through a #10 mesh tissue sieve to separate the heads, which went through the sieve, from the larger abdomen, thoraxes, and residual intact houseflies that did not pass through the sieve. Contaminating wings were removed by holding a vacuum nozzle approximately 4 cm above the heads, and contaminating legs were separated from the heads by passage through a #15 mesh screen. All remaining debris were removed from the pool of heads using forceps. The purified heads were collected in 50 mL polypropylene conical tubes and stored in liquid nitrogen until processed further.
About 13 g of purified housefly heads were suspended in about 65 mL of 10% sucrose buffer (10% sucrose (w/w) in 10 mM Tris, pH 7.5). The heads were homogenized for about 1 minute, using a Tissumizer(trademark) homogenizer equipped with a SDT-100EN probe (available from Tekmar-Dohrmann, Cincinnati, Ohio) running at 70% of its maximum speed. The extract was further homogenized by about 5 passes through a 40 mL Dounce tissue grinder. The extract was then centrifuged at about 500xc3x97g for about 5 minutes to pellet large debris. The supernatant was collected; the pellet was washed with an additional 65 mL of 10% sucrose buffer and centrifuged at 500xc3x97g for about 5 minutes. The second supernatant was collected and combined with the first supernatant, and the pool was filtered through a 100xcexc CellMicroSieve(trademark) mesh to remove residual debris (available from BioDesign of New York, Carmel, N.Y.).
Neuronal membranes containing active GABA receptors were collected via sucrose density centrifugation by the following method. About 8 mL of 35% sucrose buffer (35% sucrose (w/w) in 10 mM Tris, pH 7.5), were dispensed into each of six 38 mL ultracentrifuge tubes. These layers were overlaid with about 8 mL of 20% sucrose buffer (20% sucrose (w/w) in 10 mM Tris, pH 7.5), and finally overlaid with about 20 mL of filtered extract supernatant. The tubes were centrifuged at about 120,000xc3x97g for about 100 min at 4xc2x0 C. After centrifugation, the 10% sucrose layer and most of the 20% sucrose layer were removed by aspiration. The membranes at the interface of the 20% sucrose and 35% sucrose layers were collected, pooled, diluted with 10% sucrose buffer, and centrifuged at about 120,000xc3x97g for about 40 min at 4xc2x0 C. After centrifugation, the supernatant was discarded, and the pellets resuspended in about 6.5 mL of assay buffer (10 mM phosphate, 300 mM NaCl, pH 7.5) using a 10 mL Potter-Elvehjem tissue grinder with a Teflon(copyright) pestle. Protein concentration was determined by the Bio-Rad Protein Assay (available from Bio-Rad Laboratories, Hercules, Calif.) using bovine serum albumin as a standard. The membranes were aliquoted and stored in liquid nitrogen for up to 2 months before use.
Mouse brains were obtained from carbon dioxide-asphyxiated Swiss-Webster mice, washed with phosphate-buffered saline, and used either fresh or after storage at xe2x88x9280xc2x0 C. for up to 10 months. Unless specified, all preparation steps were performed at 0-4xc2x0 C. For each preparation, 20 brains were suspended in about 40 mL of 0.32 M sucrose and homogenized for about 2 minutes, using a Tissumizer(trademark) homogenizer equipped with a SDT-100EN probe (available from Tekmar-Dohrmann, Cincinnati, Ohio) running at 50% of its maximum speed. The extract was centrifuged for about 5 min at about 1000xc3x97g to pellet intact brain tissue. The supernatant was retained and the pellet washed with an additional 40 mL of 0.32 M sucrose and centrifuged at 1000xc3x97g for about 5 minutes. The 1000xc3x97g supernatants were combined and centrifuged at about 10,000xc3x97g for about 20 min to pellet membranes. The 10,000xc3x97g supernatant was discarded and the pellet was resuspended in about 20 mL of water containing 1 mM EDTA. The sample was dialyzed two times for about 3 hours each against about 3 L of water. The sample was then centrifuged at about 25,000xc3x97g for about 30 min to pellet the membranes. After centrifugation, the supernatant was discarded and the wet pellet recovered. The protein concentration of the pellet was determined by the Bio-Rad Protein Assay (available from Bio-Rad Laboratories, Hercules, Calif.) using bovine serum albumin as a standard. The membranes were aliquoted and stored at xe2x88x9280xc2x0 C. for up to 6 months before use.
The present invention is also directed to the multi-step synthesis of compounds of Formula I, including intermediates and intermediate reaction steps as herein described.
Compounds of the present invention can be synthesized according to methods outlined in the following scheme descriptions.
Scheme 1: 2,6-Dichloro-4-trifluoromethylphenylhydrazine was condensed with 3-cyanotetrahydrothiophen-4-one under acidic conditions to obtain aminopyrazole I which was then oxidized with m-CPBA to obtain the corresponding sulfoxide II and sulfone III. Deamination of I gave pyrazole IV, whereas acetylation of I gave the acetamide V. Sulfoxide IVxe2x80x2 and sulfone IVxe2x80x3 were obtained by oxidation of IV with m-CPBA. Likewise, sulfoxide Vxe2x80x2 and sulfone Vxe2x80x3 were obtained by oxidation of V with m-CPBA.
Scheme 2: 2,6-Dichloro-4-trifluoromethylphenylhydrazine was condensed with 3-carbomethoxytetrahydrothiophen-4-one to obtain the corresponding hydrazone IX which was cyclized under basic conditions to obtain the corresponding pyrazolone X. Pyrazolone X was also converted to the corresponding chloride XIa, phenyl sulfonate XIb, methyl ether XIc, and benzoate XId under standard conditions. Pyrazolone X and pyrazoles XIa-d were converted to the corresponding sulfoxides (n=1) and sulfones (n=2) by oxidation with m-CPBA.
Scheme 3: Cycloalkanones XII (n=0, 1 or 2) were treated with LDA and TMSCl to obtain the corresponding silylenol ethers XIII which were subsequently treated with TMSI and trimethylorthoformate to obtain the corresponding ketoacetals XIV. The ketoacetals XIV were condensed with 2,6-dichloro-4-trifluoromethylphenylhydrazine under acidic conditions to obtain the corresponding cycloalkapyrazoles XV.
Scheme 4: 2-Nitroaldehydes XVI were condensed with 2,6-dichloro-4-trifluoromethylaniline to obtain the corresponding imines XVII which were treated with (EtO)3P to arrive at the corresponding pyrazoles XVIII.
Scheme 5: Reaction of organometallic reagents with optionally substituted cyclopentenone XIX followed by enolate trapping with TMSCl/HMPA afforded the corresponding silyl enol ethers XX which were converted to the corresponding ketoacetals XXI by treatment with TMSI and trimethylorthoformate. The ketoacetals XXI were subsequently condensed with 2,6-dichloro-4-trifluoromethylphenylhydrazine to obtain the corresponding pyrazoles XXII.
Scheme 6: Optionally-substituted cycloalkanones XXIII were treated with BF3OEt2 and triethyl- or trimethylorthoformate to obtain the corresponding ketoacetals XXIV which were condensed with 2,6-dichloro-4-trifluoromethylphenylhydrazine to obtain the corresponding optionally-substituted pyrazoles XXV.
Scheme 7: The halocarboxylic acids XXVII, obtained from dihalocycloalkenes XXVI by treatment with alkyl lithium and CO2, were converted first to acyl halides, and then converted to the corresponding amides XXVIII under standard conditions. Dehydration of the amides XXVIII gave the corresponding nitriles XXIX which were condensed with 2,6-dichloro-4-trifluoromethylphenylhydrazine to obtain the corresponding amino pyrazoles XXX. Deamination of the aminopyrazoles XXX gave corresponding pyrazoles XXXI.
Scheme 8: The carboxylic acids XXXII were converted to the corresponding acid chlorides XXXIII and then reacted with the Wittig reagent. Pyrolysis gave the corresponding acrylonitriles XXXV which were condensed with 2,6-dichloro-4-trifluoromethylphenylhydrazine to obtain the corresponding pyrazoles XXXVI. The side chain halogen was then displaced by a protected thiol, e.g., tritylthiol, and the 4 position of the resulting pyrazole XXXVII was brominated under free radical conditions. Deprotection of thiol XXXVIII followed by cyclization gave the corresponding tetrahydrothienopyrazoles XL. Deamination gave the corresponding pyrazoles XLI and oxidation with m-CPBA gave the corresponding sulfoxides XLII and sulfones XLIII.
Scheme 9: Pyrazole 11 was oxidized to pyrazolone 48 and 49. The ratio of 48 to 49 formed from the reaction was dependent upon the oxidizing conditions used. Pyrazolone 48 was treated with ethanedithiol to form pyrazole 62. Pyrazole 62 was converted to difluoropyrazole 63 by treatment with N-iodosuccinimide (NIS) and pyridinium poly(hydrogen fluoride) (PPHF). Likewise, pyrazolone 48 was converted, under basic conditions, to the silyl enol ether, which was then reacted with 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate) (SELECTFLUOR) to provide fluoropyrazolone 54. Fluoropyrazolone 54 was reacted with ethanedithiol to yield fluoropyrazole 55, which was then treated with NIS and PPHF to produce trifluoropyrazole 56. Alternatively, Grignard reaction of 54 provided monofluorohydroxypyrazole 57. Pyrazolone 48 was also used to produce pyrazole 60, which was converted to difluoropyrazole 61. Pyrazolone 49 was fluorinated with diethylaminesulfur trifluoride (DAST) to produce difluoropyrazole 50. Difluoropyrazole 50 was oxidized to difluoropyrazolone 51, which was converted to difluoropyrazole 52, followed by fluorination to yield tetrafluoropyrazole 53. Additionally, pyrazolone 49 was lithiated, treated with TMSCl to form the silyl enol ether, and reacted with SELECTFLUOR to form fluoropyrazolone 58. Fluoropyrazolone 58 was converted to fluoropyrazole 59.
Scheme 10: Reduction of pyrazolone 48 yielded hydroxypyrazole 62, which was reacted with thionyl chloride to produce pyrazole 63 and chloropyrazole 64. Treatment of 64 with trifluoromethylthiocuprate provided trifluoromethylthiopyrazole 65, which was subsequently oxidized to produce sulfoxide 66 and sulfone 67. Additionally, chloropyrazole 64 was treated with methylthiocuprate to yield methylmercaptopyrazole 68, which was subsequently oxidized to produce sulfoxide 69 and sulfone 70.
Scheme 11: Difluoropyrazolone 51 was reduced to yield difluorohydroxypyrazole 75, which was methylated under basic conditions to provide difluoromethoxypyrazole 76. Additionally, difluoropyrazolone 51 was reacted with a Grignard reagent to produce difluorohydroxypyrazole 77, which was treated with DAST, yielding trifluoropyrazole 78.
Scheme 12 Pyrazole 79 was oxidized under standard conditions to yield dihydroxypyrazole 80, which was fluorinated with DAST to provide difluoropyrazole 81. Additionally, pyrazole 79 was treated with mCPBA to provide oxirane 82. Fluorination of 82 with PPHF provided fluorohydroxypyrazole 83.
Scheme 13: Alcohol 64 in benzene was heated at reflux with p-Toluenesulfonic acid (pTSA)for 6 hr to obtain 1:1 mixture of cyclopentens 65 and 83. This mixture was then converted to diols 84 and 85 by treating with OsO4 and N-methylmorpholine-N-oxide (NMO). The two diols were then separated and 84 was converted to difluo compound 86 by treating it with DAST. Compound 86 was then oxidized to ketone 87 with CrO3 and the resulting ketone was reacted with Grignard reagents to obtain tertiary alcohols 88. Compound 85 was converted to ketone 90 by heating with pTSA. The ketone 90 was reacted with ethanedithiol to obtain the corresponding thiolane which was converted to gem difluoro compound 91. Oxidation of 91 with CrO3 gave ketone 92 which was converted to tetrafluoro compound 94 via dithiolane. Reaction of ketone 94 with Grignard reagents gave alcohols 93.
The included examples are illustrative, but not limiting, of the compounds, methods and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.
Compounds of the present invention are shown in Table I and Schemes 1-13. In Schemes 9-11, Ar is equivalent to 2,6-dichloro-4-trifluoromethylphenyl radical. Compounds having other aromatic groups in the 1-position can be synthesized by substituting other known hydrazines for 2,6-dichloro-4-trifluoromethylphenyl hydrazine in the following schemes. 